Use of the Meganuclease I-SceI of Saccharomycescerevisiae to select for gene deletions in actinomycetes

Abstract

The search for new natural products is leading to the isolation of novel actinomycete species, many of which will ultimately require genetic analysis. Some of these isolates will likely exhibit low intrinsic frequencies of homologous recombination and fail to sporulate under laboratory conditions, exacerbating the construction of targeted gene deletions and replacements in genetically uncharacterised strains. To facilitate the genetic manipulation of such species, we have developed an efficient method to generate gene or gene cluster deletions in actinomycetes by homologous recombination that does not introduce any other changes to the targeted organism's genome. We have synthesised a codon optimised I-SceI gene for expression in actinomycetes that results in the production of the yeast I-SceI homing endonuclease which produces double strand breaks at a unique introduced 18 base pair recognition sequence. Only those genomes that undergo homologous recombination survive, providing a powerful selection for recombinants, approximately half of which possess the desired mutant genotype. To demonstrate the efficacy and efficiency of the system, we deleted part of the gene cluster for the red-pigmented undecylprodiginine complex of compounds in Streptomyces coelicolor M1141. We believe that the system we have developed will be broadly applicable across a wide range of actinomycetes.

Two dynamin-like proteins stabilize FtsZ rings during Streptomyces sporulation

“…The resulting mutant strains were confirmed by PCR analysis. The markerless ΔdynA (SS255) mutant strain was generated using I-SceI Meganuclease-mediated gene deletion as described by Fernández-Martínez and Bibb (45).…”

Engineering microbial hosts for production of bacterial natural products

“…36 Alternatively, exploiting the inefficient non-homologous end-joining (NHEJ) in vegetative cells and the lethality of double stranded chromosomal breaks in the absence of HR repair, the meganuclease I-SceI from Saccharomyces cerevisiae has been adapted to select for double-crossover recombinants in actinomycetes. 40, 41 In the first of this two-step method, an 18 bp I-SceI recognition site is co-introduced with a selection marker into the genome by single-crossover recombination. When I-SceI is introduced in a second step, only double-crossover recombinants (revertants or desired mutants) that have repaired the targeted chromosomal lesion created by I-SceI will survive.…”

“…This strategy has been successfully used to generate large markerless gene deletions in S. coelicolor and S. venezuelae . 40, 41 …”

Toward Systems Metabolic Engineering of Streptomycetes for Secondary Metabolites Production

“…[41] To aid genetic engineering effort even further in streptomycetes, an alternative to this classical method was reported using the S. cerevisiae I-SceI homing endonuclease for DNA double strand break (DSB)-based genome editing. [42,43] This meganuclease differs from conventional restriction enzymes by its longer recognition site of 18 bp and with a lower risk of off-targets for gene editing purposes. Using a twoplasmid system, the I-SceI recognition sequence and a suitable resistance marker are introduced into a defined site on the genome of the recipient streptomycete via a single crossover event using the first plasmid.…”